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Speckled snow mold caused by Typhula ishikariensis is one of the most devastating diseases of winter wheat in Hokkaido, Japan and parts of the Pacific Northwest region of USA. Munstertaler is a winter wheat landrace from Switzerland that has very high resistance to snow mold and superior freezing tolerance. Quantitative trait loci (QTL) for resistance to speckled snow mold were identified in a doubled haploid population derived from a cross between Munstertaler and susceptible variety Ibis, both under field conditions and controlled environment tests. Composite interval mapping analysis revealed a major QTL on chromosome 5D from Munstertaler, and on chromosome 6B from Ibis. Flanking microsatellite marker cfd 29 for the QTL on chromosome 5D was about 5 cM distant from vernalization requirement gene Vrn-D1, suggesting that the QTL on chromosome 5D is located on a cold-stress-related gene cluster along with Vrn-D1 and freezing tolerance gene Fr-D1. The QTL on chromosome 6B from Ibis was located on the centromere region flanking QTn.mst-6B, which is reported to increase plant tiller number.

Antifreeze proteins (AFPs) inhibit ice growth by adsorbing onto specific ice planes. Microbial AFPs show diverse antifreeze activity and ice plane specificity, while sharing a common molecular scaffold. To probe the molecular mechanisms responsible for AFP activity, we here characterized the antifreeze activity and crystal structure of TisAFP7 from the snow mold fungus Typhula ishikariensis. TisAFP7 exhibited intermediate activity, with the ability to bind the basal plane, compared with a hyperactive isoform TisAFP8 and a moderately active isoform TisAFP6. Analysis of the TisAFP7 crystal structure revealed a bound-water network arranged in a zigzag pattern on the surface of the protein’s ice-binding site (IBS). While the three AFP isoforms shared the water network pattern, the network on TisAFP7 IBS was not extensive, which was likely related to its intermediate activity. Analysis of the TisAFP7 crystal structure also revealed the presence of additional water molecules that form a ring-like network surrounding the hydrophobic side chain of a crucial IBS phenylalanine, which might be responsible for the increased adsorption of AFP molecule onto the basal plane. Based on these observations, we propose that the extended water network and hydrophobic hydration at IBS together determine the TisAFP activity.

In previous testing, we found that gray snow mold, caused by Typhula ishikariensis or T. incarnata, could be controlled by applying T. phacorrhiza onto turf grass prior to snowfall. To test for phytopathogenicity of this biocontrol agent, the five most disease-suppressive isolates were cultured on mixed grains and applied to 12 turfgrass cultivars and 2 winter wheat cultivars in December 1995 and 1996. After snow melt in April 1996 and 1997, significantly greater winter injury was visible on plots treated with the pathogens compared to T. phacorrhiza-treated plots or untreated plots. Except for one cultivar in 1996, there were no significant differences between T. phacorrhiza-treated plots and untreated plots. Pelletized alginate formulations of T. phacorrhiza containing kaolin clay with various nutritional amendments were tested for viability and efficacy. After 64 weeks of storage, viability remained >85% at -15°C and >70% at 4°C, but 2) was equivalent to using larger amounts of mixed grain inoculum (200 g/m2) or wheat bran inoculum (100 g/m2) in 2 years of field testing.

Snow mold fungus, Typhula ishikariensis , secretes seven antifreeze protein isoforms (denoted Tis AFPs) that assist in the survival of the mold under snow cover. Here, the X-ray crystal structure of a hyperactive isoform, Tis AFP8, at 1.0 A resolution is presented. Tis AFP8 folds into a right-handed β-helix accompanied with a long α-helix insertion. Tis AFP8 exhibited significantly high antifreeze activity that is comparable with other hyperactive AFPs, despite its close structural and sequence similarity with the moderately active isoform Tis AFP6. A series of mutations introduced into the putative ice-binding sites (IBSs) in the β-sheet and adjacent loop region reduced antifreeze activity. A double-mutant A20T/A212S, which comprises a hydrophobic patch between the β-sheet and loop region, caused the greatest depression of antifreeze activity of 75%, when compared with that of the wild-type protein. This shows that the loop region is involved in ice binding and hydrophobic residues play crucial functional roles. Additionally, bound waters around the β-sheet and loop region IBSs were organized into an ice-like network and can be divided into two groups that appear to mediate separately Tis AFP and ice. The docking model of Tis AFP8 with the basal plane via its loop region IBS reveals a better shape complementarity than that of Tis AFP6. In conclusion, we present new insights into the ice-binding mechanism of Tis AFP8 by showing that a higher hydrophobicity and better shape complementarity of its IBSs, especially the loop region, may render Tis AFP8 hyperactive to ice binding.

Speckled snow mold, caused by Typhula ishikariensis, is one of the most important Typhula snow molds in subarctic zones of the Northern Hemisphere. Nine isolates of three T. ishikariensis varieties (var. ishikariensis, var. canadensis, and var. idahoensis) isolated from infected turfgrasses on golf course fairways throughout Wisconsin were evaluated for their aggressiveness toward nine cultivars of three bentgrass species (three creeping, three colonial, and three velvet cultivars) under controlled environmental conditions. Speckled snow mold severity increased as inoculum concentration of T. ishikariensis was increased. In general, bentgrass susceptibility increased between 9 and 11 weeks after seeding but gradually decreased thereafter, suggesting expression of age-related resistance as plants matured. Significant differences in aggressiveness were detected within and among T. ishikariensis varieties. Significant interactions between T. ishikariensis varieties or isolates and bentgrass species were detected, but there was no interaction between pathogen isolates and bentgrass cultivars. Disease severity evaluations showed significant differences among bentgrass cultivars and species in their response to T. ishikariensis. Since bentgrass species exhibit differential responses to T. ishikariensis varieties, representative isolates of each variety should be employed for screening of bentgrass germplasm for resistance to speckled snow mold.

Nishio, Z., Iriki, N., Takata, K., Ito, M., Tabiki, T., and Murray, T. D. 2008. Influence of coldhardening and soil matric potential on resistance to speckled snow mold in wheat. Plant Dis. 92:1021-1025. The influence of soil matric potential, cold-hardening temperature, and duration on resistance to speckled snow mold caused by Typhula ishikariensis in wheat was investigated. Six winter wheat lines were subjected to cold-hardening temperatures of 2 or 4°C for 1, 2, 3, or 4 weeks with soil matric potential of –0.1 or –0.01 MPa. Plants were inoculated with T. ishikariensis after cold-hardening, incubated at 10°C for 25 days in the dark, and then evaluated for regrowth. Overall recovery from snow mold was least when plants were hardened at 2°C for 1 week at –0.01 MPa and greatest when hardened at 4°C for 4 weeks at –0.1 MPa. Survival of plants following snow mold was greater when plants were cold-hardened at 4 than at 2°C and at –0.1 than –0.01 MPa soil matric potential. The greatest difference in survival among lines and correlation with field observations occurred when plants were hardened at 4°C at –0.1 MPa matric potential for 3 weeks. Understanding the influence of temperature and soil matric potential during coldhardening on speckled snow mold resistance will be useful to breeding programs developing snow-mold-resistant cultivars under controlled environment conditions.

Typhula ishikariensis Imai is a causal agent of Typhula snow mold, one of the most important turfgrass diseases in northern regions of the United States. Within Wisconsin isolates, there are three district groups clustered with known isolates of T. ishikariensis var. ishikariensis, var. canadensis and var. idahoensis as identified by RAPD markers. To further investigate the genetic relationship among these groups (varieties), monokaryon-monokaryon and dikaryon-monokaryon mating experiments were conducted. Mating types from var. ishikariensis, var. canadensis and var. idahoensis isolates were paired in all possible combinations. Pairings between var. canadensis and var. idahoensis were highly compatible, while no compatibility was detected between var. ishikariensis and either var. canadensis or var. idahoensis. These results indicate that var. ishikariensis is genetically separated from var. canadensis and var. idahoensis, whereas var. canadensis and var. idahoensis appeared to be genetically related to each other as a taxonomic unit. In the genetic relationship with the known biological species, var. ishikariensis and var. canadensis were genetically related to biological species I and II, respectively. However, var. idahoensis was not compatible with any of the biological species, suggesting that the pathogen may be in the process of biological speciation from var. canadensis.

Typhula blight is a major problem on golf courses where snow cover persists for long periods. The disease is primarily managed by applying a mixture of fungicides in the autumn prior to winter snow cover. The fungicides chlorothalonil and fludioxonil are often included in these mixtures even though they do not completely suppress Typhula blight when applied alone. We studied whether minimal control was due to a lack of persistence of these fungicides under snow, or whether certain isolates of Typhula ishikariensis and T. incarnata exhibited tolerance to these fungicides. The persistence of chlorothalonil and fludioxonil residues in the turf was determined during the winters of 2005–06, 2006–07 and 2007–08. Concentrations of chlorothalonil and fludioxonil in the verdure decreased or remained nearly the same at most sampling dates, indicating these fungicides did not dissipate rapidly under snow. Despite this, only marginal control of Typhula blight was observed in the fungicide-treated plots. The i...

Organisms that live in cold climates have to confront a lot of obstacles due to ice crystal formation. As the temperature dips lower than sub-zero, ice crystals gradually develop inside the cell which leads to cell rupture. Antifreeze proteins (AFPs) are produced as a specialized adaptation by these psychrophiles that permit their survival in subzero environment. Antifreeze proteins produced by fungi are thought to bind onto ice crystals quickly using a regular array of well conserved residues/motifs and alter the structure of ice in or around the host thereby hindering growth and re-crystallization of ice. In this investigation, seven different antifreeze proteins (AFPs) of Typhula ishikariensis retrieved from Uniprot database were analyzed and characterized in silico using various computational tools. Their physico-chemical properties, hydropathicity and secondary structure have been identified. Phylogenetic analysis was also performed using MEGA4. The study might be an initiation in understanding the underlying structural and functional aspects of these proteins, their common characteristics and other features for academic and industrial purposes.

Metabolism of fructans in temperate grasses dynamically fluctuates before and during winter and is involved in the overwintering activity of plants. We monitored three candidate factors that may be involved in seasonal fructan metabolism in timothy (Phleum pratense): transcription levels of two fructosyltransferase (PpFT1 and PpFT2) genes and one fructan exohydrolase (Pp6-FEH1) gene during fall and winter and under artificially cold conditions. Functional analysis using a recombinant enzyme for PpFT2, a novel fructosyltransferase cDNA, revealed that it encoded sucrose:fructan 6-fructosyltransferase, with enzymatic properties different from previously characterized PpFT1. PpFT1 transcripts decreased from September to December as the amount of fructans increased, whereas PpFT2 transcripts increased in timothy crowns. PpFT2 was transcriptionally more induced than PpFT1 in response to cold and sucrose in timothy seedlings. A rapid increase in Pp6-FEH1 transcripts and increased monosaccharide content were observed in timothy crowns when air temperature was continuously below 0°C and plants were not covered by snow. Transcriptional induction of Pp6-FEH1 by exposure to -3°C was also observed in seedlings. These findings suggest Pp6-FEH1 involvement in the second phase of hardening. PpFT1 and PpFT2 transcription levels decreased under snow cover, whereas Pp6-FEH1 transcription levels were constant, which corresponded with the fluctuation of fructosyltransferase and fructan exohydrolase activities. Inoculation with snow mold fungi (Typhula ishikariensis) increased Pp6-FEH1 transcription levels and accelerated hydrolysis of fructans. These results suggest that transcriptional regulation of genes coding fructan metabolizing enzymes is partially involved in the fluctuation of fructan metabolism during cold acclimation and overwintering.

Snow moulds are fungi and fungal-like microbes that occur frequently as pathogens of moss and vascular plant species in the Arctic and Antarctic, chiefly in maritime areas with permanent snow cover for several months of each year. Here, we review the environments inhabited by polar snow moulds, their distribution and the macroscopic features of infections, such as the sclerotia that form on the leaves of higher plants, or the more frequently encountered concentric rings found in moss stands. The microscopic features of infections are described, as are the taxa of snow moulds found in polar habitats, such as the ascomycetes Thyronectria antarctica var. hyperantarctica and Sclerotinia borealis, the basidiomycete Typhula ishikariensis and the oomycete Pythium. Recent research, also reviewed here, indicates that a heterothallic species of Pythium apparently has a bipolar distribution. The adaptations of snow moulds to polar environments, such as their growth at low temperatures, are covered, as are avenues for future research on these microbes.

Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis . Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

Despite their close association with human activities, plant pathogenic fungi have rarely been found in archaeological excavations. We report here that a fungus was closely associated with human activities even in prehistoric times. Sclerotium-like objects were found at historical sites (4000 to 400 BP) on the island of Hokkaido, northern Japan. They were spherical, 0.3-1.0 mm in diameter, and had a medulla and rind. Some had leaf fragments on the surface or a protuberance that resembled emerging sporocarp primordia. These traits indicated that they were sclerotia of the snow mold fungus, Typhula ishikariensis biotype B.

Antifreeze proteins are structurally diverse polypeptides that have thermal hysteresis activity and have been discovered in many cold-adapted organisms. Of these, fungal antifreeze protein has been purified and partially characterized only in a species of psychrophilic basidiomycete, Typhula ishikariensis. Here we report a new fungal antifreeze protein from another psychrophile, Antarctomyces psychrotrophicus. We examined its biochemical properties and thermal hysteresis activity, and compared them with those of the T. ishikariensis antifreeze protein. The antifreeze protein from A. psychrotrophicus was purified and identified as an extracellular protein of approximately 28 kDa, which halved in size following digestion with glycosidase. The A. psychrotrophicus antifreeze protein generated bipyramidal ice crystals and exhibited thermal hysteresis activity (for example thermal hysteresis = 0.42 °C for a 0.48 mm solution) similar to that of fish antifreeze proteins, while a unique rugged pattern was created on the facets of the ice bipyramid. The thermal hysteresis activity of the A. psychrotrophicus antifreeze protein was maximized under alkaline conditions, while that of the T. ishikariensis antifreeze protein was greatest under acidic conditions. The T. ishikariensis antifreeze protein exhibited a bursting ice growth normal to the c-axis of the ice crystal and high thermal hysteresis activity (approximately 2 °C), as in the case of insect hyperactive antifreeze proteins. From these results, we speculate that the A. psychrotrophicus antifreeze protein is very different from the T. ishikariensis antifreeze protein, and that these two psychrophiles have evolved from different genes.

Two Gram-variable, aerobic, motile, rod-shaped, endospore-forming bacterial strains, M4-2T and M4-1, were isolated from soil samples collected from Oblast Magadan, Russian Far East, as micro-organisms antagonistic to the psychrophilic phytopathogenic fungus Typhula ishikariensis. Strains M4-2T and M4-1 were identified as members of the genus Paenibacillus by phenotypic and phylogenetic analyses based on 16S rRNA gene sequences. The strains contained anteiso-C15:0 as the major fatty acid (63.0-64.7%) and MK-7 as the major isoprenoid quinone. The DNA G+C contents were 42.8 and 41.7 mol%, respectively. 16S rRNA gene sequence analysis showed that strains M4-2T and M4-1 exhibited high similarities with Paenibacillus macquariensis DSM 2T (99.5 and 99.7%, respectively) and Paenibacillus antarcticus LMG 22078T (99.4 and 99.5%, respectively). There were no clear differences in the phenotypic characteristics and chemotaxonomic and phylogenetic data between the novel isolates and P. macquariensis DSM 2T. DNA-DNA hybridization experiments between strain M4-2T and P. macquariensis DSM 2T and P. antarcticus LMG 22078T revealed reassociation values of 56 and 49%, respectively. Multilocus sequence analysis confirmed the differences between the new isolates and reference strains that were observed with the DNA-DNA hybridization studies. On the basis of the results described above, it is proposed that the isolates represent a novel subspecies of P. macquariensis, Paenibacillus macquariensis subsp. defensor subsp. nov. The type strain is M4-2T (=JCM 14954T=NCIMB 14397T).

Fungi have developed a variety of mechanisms for tolerating cold, including production of proteins that bind to ice, as shown by their ability to slightly lower the freezing point. At present, only one of these proteins, from the snow mold Typhula ishikariensis, and partial transcripts of a similar protein from shiitake mushroom, Lentinula edodes, have been identified. Here, we report the full sequences of ice-binding proteins from shiitake and another mushroom, the cold-adapted Flammulina populicola (enoki mushroom), and show that the recombinant proteins have ice-binding activity. The three proteins share 50-55% identities and are similar to other ice-binding proteins recently identified in ice bacteria and sea ice diatoms. The possibility that ice-binding protein genes have spread among these phyla by horizontal transfer is discussed.

Hoshino, T., Tronsmo, A.M. & Yumoto, I. 2008. Snow mold fungus, Typhula ishikariensis group III, in Arctic Norway can grow at a sub-lethal temperature after freezing stress and during flooding. – Sommerfeltia 31: 125-131. ISBN 82-7420-045-4. ISSN 0800-6865. Isolates of the snow mold fungus Typhula ishikariensis group III, which is predominant in Finnmark (northern Norway) and Svalbard, are more resistant to freezing stress than group I isolates from the southern part of Norway. Group III isolates showed irregular growth on potato dextrose agar (PDA) plates when subjected to heat stress at 10˚C. However, group III isolates showed relatively good growth on PDA at 10˚C after freezing treatment. The optimal temperatures for mycelial growth were 5˚C on PDA and 10˚C in potato dextrose broth (PDB), and group III isolates showed normal mycelial growth at 10˚C in PDB. Mycelium of group III isolates cultivated in water poured into PDA plates, and normal hyphal extension was observed only in the liquid media. Hyphal growth became irregular when mycelia had extended above the surface of the liquid media. These results suggested that group III isolates can grow at a sub-lethal temperature after freezing stress and during flooding. Soil freezing and thawing occurs regularly in the Arctic, and physiological characteristics of group III isolates are well adapted to climatic conditions in the Arctic.

Accumulation of soluble carbohydrates plays an important role in enhancement of resistance to freezing and snow mold of plants during cold acclimation. Nevertheless, few studies have examined whether changes in cell wall properties are involved in enhancement of resistance during cold acclimation. In this study, four winter wheat cultivars were sown in a field on six different dates during August-October, and their resistance to freezing and snow mold were compared in relation to soluble carbohydrate content and cell-wall mass in leaves. Resistance to freezing and snow mold was much higher in the plants sown on 23 September than in those sown on 9 September. The percentage of cell-wall mass in leaf to total dry mass (%CW) and water-soluble carbohydrate content also increased considerably during 9-23 September. Multiple regression analyses revealed that %CW contributed significantly to freezing resistance, whereas total water-soluble carbohydrate content contributed significantly to snow mold resistance. These results suggest that increased %CW enhances freezing resistance during cold acclimation.

TAD1 (Triticum aestivum defensin 1) is induced during cold acclimation in winter wheat and encodes a plant defensin with antimicrobial activity. In this study, we demonstrated that recombinant TAD1 protein inhibits hyphal growth of the snow mold fungus, Typhula ishikariensis in vitro. Transgenic wheat plants overexpressing TAD1 were created and tested for resistance against T. ishikariensis. Leaf inoculation assays revealed that overexpression of TAD1 confers resistance against the snow mold. In addition, the TAD1-overexpressors showed resistance against Fusarium graminearum, which causes Fusarium head blight, a devastating disease in wheat and barley. These results indicate that TAD1 is a candidate gene to improve resistance against multiple fungal diseases in cereal crops.

Numerous container-based studies in greenhouses or growth rooms have consistently demonstrated a suppressive effect of composts on soil-borne diseases such as damping-off and root rots (Pythium ultimum, Rhizoctonia solani, Phytophthora spp.), and wilts (Fusarium oxysporum and Verticillium dahliae). Composts have also been shown to suppress several diseases in the field, although the effects have been generally smaller and more variable than in container experiments. Several diseases of turf grass were suppressed by top-dressing with compost. These diseases are Fusarium patch (Microdochium nivale), red thread (Laetisaria fuciformis), damping-off (Pythium graminicola), brown patch (Rhizoctonia solani), dollar spot (Sclerotinia homoeocarpa) and snow mould (Typhula ishikariensis). The disease suppressive effect of compost generally increased with rate of application. Compost inclusion rates of at least 20% (v/v) were normally required to consistently obtain a disease suppressive effect, particularly in peat-b...

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Two composts were evaluated for suppression of Fusarium Patch (Microdochium nivale) and Typhula Blight (Typhula ishikariensis) snow molds in field experiments, conducted in 1998–1999 in two distinct areas of turf management. One plot was on a creeping bentgrass green (mowing height 4 mm) and the second plot was a creeping bentgrass range (mowing height 25 mm). Fall applications of compost applied at either 48.7 or 97.4 kg /100 m 2 reduced snow mold severity to levels not significantly different (P

Composting is the degradation of organic materials through the activities of diverse microorganisms. This research examined microbial community dynamics, population levels and identification of bacteria throughout the composting process and in storage. In addition, an evaluation was performed to determine the potential for dominant bacterial isolates to suppress selected turfgrass pathogens: Sclerotinia homoeocarpa, Pythium graminicola, Typhula ishikariensis, and Microdochium nivale, responsible for causing the turfgrass diseases dollar spot, pythium blight, typhula blight, and fusarium patch, respectively. Composts supported high population levels of bacteria with 78% of cultures tested being Gram-negative. Proteolytic activity, found in 29% of cultures tested is a potential mechanism of suppression or competition with other microorganisms. Although the Biolog system did not identify a wide range of bacteria, the main Gram-negative genera identified in mature compost were Pseudomonas (28%), Serratia (20%), Klebsiella (11%), and Enterobacter (5%). Twenty-one percent of isolates tested were not identified by Biolog, and many more had similarity indexes < 0.50. The microbial identification system, based on whole cell fatty acid analysis, identified a wide range of bacteria, with a higher proportion of similarities than the Biolog system. Genera common to both testing procedures included Pseudomonas, Serratia, and Enterobacter. All Gram-positives were identified as Bacillus spp. Phospholipid fatty acid analysis, used to estimate the diversity of microbial communities, was useful in monitoring changes in microbial population in storage and during composting, as well as estimating levels of compost maturity. Plate challenge experiments revealed a number of cultures with antagonistic activity against turfgrass pathogens. There were 52, 31, 32 and 19% of the bacterial isolates tested that exhibited antagonistic activity against S. homoeocarpa, P. graminicola, T. ishikariensis, and M. nivale, respectively. Improved understanding of microbial populations and their dynamics in composts will expand their potential to act as suppressants on pathogenic fungi or turfgrass.

Early seeding and accumulation of soluble carbohydrates is critical for the full expression of snow mold [caused principally by Typhula incarnata Lasch ex. Fr., Typhula ishikariensis Imai, Sclerotinia borealis Bub. & Vleug., Microdochium nivalis (Ces, ex Berl. & Vogl.) Samuels and Hallet] resistance in winter wheat (Triticum aestivum L. em Thell). Experiments were conducted during two growing seasons in the field at Lethbridge, AB, to study how early seeding affects the quantity of simple sugars and fructan, and degree of fructan polymerization in both resistant and susceptible cultivars. Different planting dates were employed to obtain plants at different developmental stages prior to winter dormancy. Leaf and crown tissue samples were collected from 14 winter wheat cultivars differing in snow mold resistance throughout the autumn, winter, and early spring. Snow mold resistant cultivars accumulated moderate levels of simple sugars and high levels of fructans across seeding dates and maintained a higher degree of polymerization of fructans compared with snow mold susceptible cultivars. Early seeded treatments generally accumulated lower levels of simple sugars and higher levels of more highly polymerized fructan in the autumn and winter than did late seeded treatments. The closest correlations between snow mold resistance and fructan content (r = 0.87) or degree of polymerization (r = 0.74) were observed in the later seeded treatments, suggesting that early seeding masked the expression of genotypic snow mold resistance. These results demonstrate an association between early seeding and fructan accumulation in relation to snow mold resistance in winter wheat and provide a physiological basis for the higher level of snow mold resistance among early seeded treatments.

Four hundred and eighty one accessions of species and subspecies of genera Triticum and Aegilops were evaluated for resistance to speckled snow mold caused by Typhula ishikariensis and for freezing tolerance. All Triticum and seven of Aegilops species were severely affected by snow mold. Among surviving Aegilops spp., only the Ae. cylindrica accessions exhibited resistance similar to that of the most resistant winter wheat cultivar, PI 173438. After repeated screening of accessions of Ae. cylindrica, 12 accessions were identified as having resistance similar to that of PI 173438; eleven of those were considered more freezing tolerant than PI 173438, but less than wheat cv. Valjevskaya, the freezing tolerant check. Accessions of Ae. cylindrica with snow mold resistance exceeding that of PI 173438, and with freezing tolerance, are currently being screened.

Pretreatment with sodium alginate (20 g/m2) prior to application of a biocontrol agent, Pseudomonas fluorescens strain A11RN, enhanced the control turfgrass snow mold caused by Typhula ishikariensis biotype B. Disease severities in two fields relative to untreated control plots were 53% and 61% in plots treated with both the strain and sodium alginate, which were significantly lower than 69% and 77% in plots treated only with the strain. The antagonist population remained above the threshold for effective control (ca. 1×107 cfu/cm3) at least for the first month under snow in pretreatment with sodium alginate. The population of the biocontrol agent after snowmelt on turfgrass was five times higher in sodium alginate-amended plots than in unamended plots. Sodium alginate alone did not reduce disease severity. Sodium alginate might have supported the survival of the applied strain.

The distribution pattern and frequency of isolates of a snow mold fungus, Typhula ishikariensis biotype A belonging to a predominant MCG (mycelial compatibility group, referred to as super MCG), were surveyed throughout its habitat from northern Honshu to eastern Hokkaido. About 38 and 14% of isolates examined belonged to super MCG in eastern and central Hokkaido, respectively ; however, super MCG was never found in southern Hokkaido or northern Honshu. These findings imply that T. ishikariensis biotype A consists of two populations in Japan, i.e., one that is distributed in Honshu and southern Hokkaido and lacks super MCG isolates, and the other that includes super MCG isolates and exists in central and eastern Hokkaido. The difference in distribution pattern of the two populations is discussed in terms of geological history during the Pleistocene (2 million to 10 thousand years ago). The tendency of global warming, which alleviates freezing damage, was considered to be responsible for the outbreak of this fungus in eastern Hokkaido.

Isolates of the biocontrol fungus Typhula phacorrhiza were evaluated in field tests over a 5-year period for suppression of grey snow mold caused by Typhula incarnata and Typhula ishikariensis. Of the hundreds of T. phacorrhiza isolates collected from across southern Ontario in the spring of 1994, 42 were cultured on mixed grains and applied in the late fall to creeping bentgrass (Agrostis palustris) at a rate of 200 g/m2 (4 x 105 cfu/m2) along with inocula of T. incarnata or T. ishikariensis at 10 g/m2 (2 x 104 cfu/m2). In late fall of each year from 1995 to 1998, T. incarnata and T. ishikariensis were reapplied to the same plots, but not T. phacorrhiza. Plots were rated for disease after snowmelt in each year from 1995 to 1999. During the first three years, high positive correlations of winter injury were found between the suppression trials, with several isolates showing significant control of grey snow mold. By the fourth year after inoculation, suppression of snow mold in plots treated with the most ...

The mycelial growth, sclerotial production, and carbon utilization of the snow mould biocontrol agent Typhula phacorrhiza Fries were compared with the two grey snow mould fungi, Typhula ishikariensis Imai and Typhula incarnata Lasch ex Fries. Variation was observed among the four isolates for each species, but there was greater variation among species. All three species were able to grow at the lowest temperature (0°C), but temperature optima differed with T. ishikariensis lowest and T. phacorrhiza highest. On potato dextrose agar or potato malt agar at 10°C, T. phacorrhiza had greater radial growth than T. ishikariensis but less than T. incarnata. All species could utilize microcrystalline cellulose, bacto-cellulose, and glucose as carbon sources, but radial growth of T. phacorrhiza was significantly greater than T. incarnata and T. ishikariensis on these defined carbon sources tested, except for Indulin-AT, which was inhibitory to T. incarnata and T. phacorrhiza. This greater ability to utilize these structural and storage carbohydrates, combined with mycelial growth and sclerotial production over a wider range of temperatures, may help explain how some isolates of T. phacorrhiza are able to outcompete grey snow mould in field tests.Key words: turfgrass disease, biocontrol, psychrophilic.

Isolates of the fungus Typhula phacorrhiza Fries (TP) were evaluated in field tests over a 3-year period for suppression of grey snow mould caused by Typhula ishikariensis Imai (Tish) and Typhula incarnata Lasch ex Fr. (Tinc). Isolates of TP were collected across southern Ontario in the spring of 1994. In December 1994, 46 of these isolates, which had been cultured on mixed grains, were applied to creeping bent grass (Agrostis stolonifera L.) at a rate of 200 g/m2 (4 × 105 colony forming units (cfu)/m2) with inoculum of Tish or Tinc at 10 g/m2 (2 × 104 cfu/m2). In December 1995, 30 selected TP isolates were inoculated onto a new set of plots along with grey snow mould fungi. In November 1996, 22 of these isolates were re-inoculated onto the 1995 plots. All plots were rated for injury after snowmelt, 1995-1997. Isolates of TP varied significantly in their ability to suppress disease. No strong correlations were found between in vitro growth characteristics and field performance; however, significant positive correlations were found between the disease suppression trials for the 3 years, with several isolates showing statistically significant control of grey snow mould equal to a fungicide treatment.Key words: biocontrol, turfgrass disease, fungi.

Frost tolerance was determined in Norwegian varieties of groups I and III of the psychrophihc fungus Typhula mhikanensis. When myceha of group I and III strains were rapidly frozen to -40℃ in a program freezer at a cooling rate of 20℃/h, the regrowth of group I strains was delayed at their optimal growth temperature (10℃), whereas the regrowth of group III strains was not affected at their optimal growth temperature (2℃). Furthermore, when group I and III strains were rapidly frozen and regrew at 0℃, the average temperature during snowfall in the northernmost area of Norway, group I strains, in contrast to group III strains, did not regrow at all. These results suggest that group III strains are more resistant than group I strains to rapid freezing Group III strains are well adapted to climatic conditions in northernmost Norway Freezing points of myceha of group III strains were determined by calonmetenc analysis Mycelia of group III strains froze at temperatures higher than -10℃ Therefore, the freezing resistance of group III strains might be based on extracellular ice formation. In the cytosol of group III, a 30 kDa protein that strongly immunoreacted with the anti-antifreeze protein (AFP) I antibody was found, and the 30 kDa protein level in group III strains was higher than that in group I strains. These results suggest that the difference in the distribution pattern of both groups can be ascribed, at least partially, to the difference in freezing resistance of myceha.

Growth of two groups of strains (group I and III) of the snow mould fungus, Typhula ishikariensis from northern Norway, were studied in agar and liquid culture. All strains could grow from—5 to 15 (group III) or 20°C (group I) and the optimum temperatures for growth were 4 or 10°C, respectively. The group III strains showed irregular growth as the hypal extensions were inhibited at 15°C. Different temperature two‐dimensional electrophoresis revealed that in the group III strains some intracellular proteins were modified at the highest temperature. One of the reasons that group III strains did not grow or showed irregular growth at 20 or 15°C, was probably due to protein aggregation or dissociation and thereby loss of vital properties of intracellular proteins.

An extracellular lipolytic enzyme active at low temperature was purified from the culture filtrate of the snow mold fungus, Typhula ishikariensis group III, strain 6-1-1. The molecular mass of enzyme was approximately 83 kDa (SDS-PAGE). The lipolytic enzyme was most active for p-nitrophenyl palmitate at 30 –C (pH 9.0). The lipolytic activity was 23.4% of the maximum at 4 –C, the temperature of culture. Thus, the isolated T. ishikariensis lipolytic enzyme is thought to represent a new member of a group of enzymes active at low temperature.

Antifreeze proteins (AFPs) are proteins that have the ability to bind to ice crystals and inhibit the growth of ice. It is generally accepted that AFPs may protect cell membranes from freezing injury via the inhibition of ice recrystallization, thereby increasing the survival of psychrophilic microorganism at sub-zero temperatures. Screening of antifreeze activity showed that the occurrence of yeast AFPs was limited to basidiomycetous psychrophilic yeasts living under the thermal fluctuations. Currently only three species, Glaciozyma antarctica, Glaciozyma sp. AY30, and Rhodotorula glacialis are known to have AFPs or antifreeze activities. Of ten AFPs whose amino acid sequences are known, two AFPs are relatively well studied: AFP1 from G. antarctica and LeIBP from Glaciozyma sp. AY30. The amino acid sequence analysis showed that they are closely related with the fungus Typhula ishikariensis AFP, which clustered with other fungal, diatom, and bacterial AFPs to form new class of AFPs. The structural investigation of LeIBP revealed that it has an ice-binding motif (T-X-T motif) similar to those of fungal and insect AFPs. Real-time PCR of Glaciozyma AFPs exhibited that their expression is regulated by thermal fluctuation.

The Antarctic sea ice diatom Navicular glaciei produced ice-binding protein (NagIBP) that is similar to the antifreeze protein (TisAFP) from snow mold Typhula ishikariensis . In the thermal hysteresis range of NagIBP, ice growth was completely inhibited. At the freezing point, the ice grew in a burst to 6 direction perdicular to the c- axis of ice crystal. This burst pattern is similar to TisAFP and other hyperactive AFPs. The thermal hysteresis of NagIBP and TisAFP could be increased by decreasing a cooling rate to allow more time for the proteins to bind ice. This suggests the possible second binding of proteins occurs on the ice surface, which might increase the hysteresises to a sufficient level to prevent freezing of the brine pockets which habitat of N. glaciei . The secondary ice binding was described as that after AFP molecules bind onto the flat ice plane irreversibly, which was based on adsorption–inhibition mechanism model at the ice–water interface, convex ice front was formed and overgrew during normal TH measurement (no annealing) until uncontrolled growth at the nonequilibrium freezing point. The results suggested that NagIBP is a hyperactive AFP that is expressed for freezing avoidance.

Vegetative compatibility groups of Typhula ishikariensis biotype A include different isolates from various localities, and the most prevalent one (super vegetative compatibility group) was studied to reveal that this super group consisted of several genets existing throughout the habitat of T. ishikariensis biotype A in Hokkaido, Japan. Random amplified polymorphic DNA and mating incompatibility alleles were studied for 10 isolates belonging to the super vegetative compatibility group from two localities 250 km distant from each other and for five isolates belonging to different groups. Seven primers, which distinguished vegetative compatibility groups, were screened out of 60 and produced a total of 55 bands. The 10 super group isolates were divided into 5 subgroups, and the five unique isolates did not cluster with each other. Protoplasts were produced from each isolate to obtain monokaryons. They were then mated with tester monokaryons derived from basidiospores of two parental isolates, one of which belonged to the super vegetative compatibility group. Mating patterns suggested that the 10 super group isolates shared common mating incompatibility alleles but this was not the case with those belonging to unique groups. The super vegetative compatibility group was found to consist of several genets with many ramets distributed throughout the habitat of T. ishikariensis biotype A in Hokkaido. These genets were regarded as sib-related dikaryons derived from basidiospores. Keywords: Typhula ishikariensis, snow mold, genet, RAPD, mating incompatibility.

Isolates ofTyphula ishikariensis, a snow mold fungus, were collected from five localities in Norway. They were divided into three groups according to genetics, cultural morphology, etc. Group I grew normally at 10 °C. Its mating patterns with Japanese taxa were variable: compatible with both biotypes A and B; compatible with biotype A but incompatible with biotype B; and incompatible with both biotypes. Group I was prevalent in southern inland districts such as Buskerud, Oppland, and Hedmark. Group II had smaller sclerotia as compared to other groups, and its sclerotia were often covered with white mycelium in nature and in culture. Group II was compatible with biotype B only. Group III was characterized by irregular growth at 10 ° C and genetic incompatibility with biotypes A and B. Cultural morphology of group III resembled that of group I at 0 °C. Rind cell patterns of sclerotia did not separate these two groups or biotypes. Isolates of groups II and III were often obtained from coastal regions in Finmark. Mating reactions were variable: monokaryons were compatible with their respective dikaryons, and monokaryons of groups II and III occasionally mated with group I dikaryons. Dikaryons of groups II and III, however did dikaryotize monokaryons of other groups. Norwegian isolates ofT. ishikariensis were highly variable, and the orthodox nomenclature system seemed inapplicalbe at the infraspecific level.